Inoculants Including Bacillus Bacteria for Inducing Production of Volatile Organic Compounds in Plants

ABSTRACT

Disclosed are inoculants that include  Bacillus  bacteria and induce production of volatile organic compounds (VOCs) by a plant that has been treated with the inoculant.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority under 35 U.S.C.§119(e) to U.S. provisional application No. 61/421,979, filed on Dec.10, 2010, the content of which is incorporated herein by reference inits entirety.

FIELD

The present subject matter relates to the field of plantgrowth-promoting rhizobacteria (PGPR). In particular, the presentsubject matter relates to PGPR that induce production of volatileorganic compounds by plants that have been treated with the bacteria.

BACKGROUND

The induction of volatile organic compounds (VOCs) in plants has gonevirtually unexamined, despite evidence that induction of plant volatilesis dependent on the interactions of biotic factors, such as planthormones (de Bruxelles and Roberts, 2001; Ament et al., 2004),herbivore-derived elicitors (Spiteller and Boland 2003), and associatedmicroorganisms including pathogens (Preston et al., 1999; Cardoza etal., 2002), as well as abiotic factors, such as wounding (Mithofer etal., 2005), heavy metals (Mithofer et al., 2004), and temperature andlight (Takabayashi et al., 1994). Plant growth promoting rhizobacteria(PGPR) represent a wide range of root-colonizing bacteria whoseapplication often is associated with increased rates of plant growth(Kloepper, 1992; Zehnder et al., 1997), suppression of soil pathogens(Schippers et al., 1987), and the induction of systemic resistanceagainst insect pests (Kloepper et al., 1999; Ryu et al., 2004). The lackof research on induction of VOCs in plants and whether PGPR caninfluence production of VOCs in plants is surprising given that PGPR areincreasingly being applied in the production of several field crops insome parts of the world (Backman et al., 1997; Cleyet-Marcel et al.,2001). Backman et al. (1997) reported that 60-75% of the US cotton cropis treated with the PGPR product Kodiak®, a Bacillus subtilis productused for suppression of Fusarium and Rhizoctonia soil pathogens. Here,the potential effects of PGPR on induction of cotton volatiles andconsequences for attraction cotton herbivores and their parasitoids werestudied. Surprisingly, PGPR were observed to elicit changes in plantVOC's with important ramifications. Knowledge of the effects of PGPR onthe induction of plant volatiles and insect-plant interactions willlikely contribute to the increased adoption of PGPR products anddevelopment of better products and also mitigate against potentialnegative impacts of these products.

SUMMARY

Disclosed are isolated plant growth promoting rhizobacteria (PGPR) andinoculants thereof that induce production of one or more volatileorganic compounds (VOCs) by a plant that has been treated with the PGPR.Suitable PGPR may include Bacillus species.

The VOCs produced by the plant may include, but are not limited to,compounds selected from alpha-pinene, beta-pinene, beta-myrcene,cis-3-hexenyl acetate, limonene, beta-ocimene, linalool,(E)-4,8-dimethyl-1,3,7-nonatriene, methyl salicylate, decanal,cis-jasmone, caryophyllene, alpha-humulene, beta-farnesene, and mixturesthereof. The VOCs produced by the PGPR-treated plants preferably modifythe behavior of insects exposed to the VOCs. In some embodiments, theinsect is an herbivore and the VOCs reduce egg-laying or feeding of theinsect on the plant. In further embodiments, the insect is a predator orparasitoid and the VOCs attract the predator or parasitoid to the plant.

The PGPR may be a single strain, species, or genus of bacteria or maycomprise a mixture of bacterial strains, species, or genera. Forexample, the PGPR may be selected from genera including, but not limitedto, Actinobacter, Alcaligenes, Bacillus, Burkholderia, Buttiauxella,Enterobacter, Klebsiella, Kluyvera, Pseudomonas, Rahnella, Ralstonia,Rhizobium, Serratia, Stenotrophomonas, Paenibacillus, andLysinibacillus.

The PGPR may include Bacillus bacteria. The Bacillus bacteria may have acomprise a 16S rDNA nucleic acid sequence comprising SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, or SEQ ID NO:4 or may comprise a 16S rDNA nucleicacid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity to one or more of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. Specific Bacillusbacteria may include Bacillus amyloliquefaciens (e.g. Bacillusamyloliquefaciens strain AP-136, deposited with the United StatesDepartment of Agriculture on Dec. 2, 2011, under Accession No. NRRLB-50614; Bacillus amyloliquefaciens strain AP-188, deposited with theUnited States Department of Agriculture on Dec. 2, 2011, under AccessionNo. NRRL B-50615; Bacillus amyloliquefaciens strain AP-218, depositedwith the United States Department of Agriculture on Dec. 2, 2011, underAccession No. NRRL B-50618; Bacillus amyloliquefaciens strain AP-219,deposited with the United States Department of Agriculture on Dec. 2,2011, under Accession No. NRRL B-50619; and Bacillus amyloliquefaciensstrain AP-295, deposited with the United States Department ofAgriculture on Dec. 2, 2011, under Accession No. NRRL B-50620); Bacillusmojavensis (e.g. Bacillus mojavensis strain AP-209, deposited with theUnited States Department of Agriculture on Dec. 2, 2011, under AccessionNo. NRRL B-50616); Bacillus solisalsi (e.g., Bacillus solisalsi strainAP-217, deposited with the United States Department of Agriculture onDec. 2, 2011, under Accession No. NRRL B-50617); Bacillus pumilus (e.g.,Bacillus pumilus strain INR-7 (otherwise referred to as BU F-22,deposited with the United States Department of Agriculture on Jul. 23,2008, under Accession No. NRRL B-50153; and BU-F33, deposited with theUnited States Department of Agriculture on Oct. 15, 2008, underAccession No. NRRL B-50185)); Bacillus simplex (e.g., Bacillus simplexstrain ABU 288, deposited with the United States Department ofAgriculture on Feb. 18, 2010, under Accession No. NRRL B-50340); andBacillus subtilis (Bacillus subtilis strain MBI 600), deposited with theUnited States Department of Agriculture on Nov. 14, 2011, underAccession No. NRRL B-50595), and mixtures or blends thereof.

Also disclosed are inoculants that include the presently disclosed PGPRand optionally a carrier. The inoculants may comprise additional activeingredients such as phytohormones (e.g., acetoin, 2,3-butanediol, andindole-acetic acid) and anti-microbial compounds (e.g., phenylethanoland 4-hydroxybenzoate).

The disclosed PGPR and inoculants thereof may be utilized in methods formodifying insect behavior towards a plant. In some embodiments themethods include administering an inoculant comprising the PGPR to aplant, to seeds, tubers, or rhizomes of a plant, or to soil or theenvironment surrounding a plant. The method may result in reducingegg-laying or feeding of an herbivore on the plant and/or may result inattracting predators or parasitoids to the plant. Suitable plants forthe methods may include, but are not limited to alfalfa, rice, barley,rye, cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea,lentil chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip,turnip, cauliflower, broccoli, turnip, radish, spinach, onion, garlic,eggplant pepper, celery, carrot, squash, pumpkin, zucchini, cucumber,apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple,soybean, canola, oil seed rape, spring wheat, winter wheat, tobacco,tomato, sorghum, and sugarcane.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Chromatographic profiles of headspace volatiles from untreated(control) cotton plants vs. cotton plants treated with PGPR strainINR-7, PGPR Blend 8, or PGPR Blend 9. Identified compounds: (1)α-pinene; (2) β-pinene; (3) β-myrcene; (4) cis-3-hexenyl acetate; (5)Limonene; (6) β-ocimene; (7) linalool; (8) unknown; (9) caryophyllene;(10) α-humulene; (11) β-farnesene

FIG. 2. Chromatographic profiles of headspace volatiles collected fromuntreated (control 1) cotton plants uninfested with caterpillars,untreated (control 2) cotton plants infested with caterpillars, PGPRBlend 9 treated cotton plants uninfested with caterpillars, and PGPRBlend 9 treated cotton plants infested with caterpillars. Identifiedcompounds: (1) cis-3-hexenal; (2) trans-2-hexenal; (3) cis-3-hexen-1-ol;(4) trans-2-hexen-1-ol; (5) α-pinene; (6) β-pinene; (7) myrcene; (8)cis-3-hexenyl acetate; (9) trans-2-hexenyl acetate; (10) limonene; (11)β-ocimene; (12) linalool; (13) unknown; (14)(E)-4,8-dimethyl-1,3,7-nonatriene; (15) cis-3-hexenyl butyrate; (16)trans-2-hexenyl butyrate; (17) n-decanal (18) cis-3-hexenyl-2-methylbutyrate; (19) trans-2-hexenyl-2-methyl butyrate; (20) indole; (21)isobutyl tiglate; (22) (E)-2-hexenyl tiglate; (23) cis-jasmone; (24)caryophyllene; (25) α-trans bergamotene; (26) α-farnesene; (27)α-humulene; (28) β-farnesene.

FIG. 3. Root surface area (cm²) of untreated (control) cotton plants vs.cotton plants treated with PGPR strain INR-7, PGPR Blend 8, or PGPRBlend 9. Means followed by different letters are significantly different(P<0.05, ANOVA, Tukey-Kramer HSD multiple comparison test, n=8)

FIG. 4. Root volume (cm³) of untreated (control) cotton plants vs.cotton plants treated with PGPR strain INR-7, PGPR Blend 8, or PGPRBlend 9. Means followed by different letters are significantly different(P<0.05, ANOVA, Tukey-Kramer HSD multiple comparison test, n=8).

FIG. 5. Root dry weight (g) of untreated (control) cotton plants vs.cotton plants treated with PGPR strain INR-7, PGPR Blend 8, or PGPRBlend 9. Means followed by different letters are significantly different(P<0.05, ANOVA, Tukey-Kramer HSD multiple comparison test, n=8)

FIG. 6. Response of naïve female M. croceipes in a four-choiceolfactometer to untreated (control) cotton plants vs. cotton plantstreated with PGPR strain INR-7, PGPR Blend 9, or blank control (emptychamber). Thirty-two parasitoids were tested each day and replicatedfive times. Means followed by different letters are significantlydifferent (P<0.05, ANOVA, Tukey-Kramer HSD multiple comparison test,n=5)

FIG. 7. Responses of naïve female M. croceipes in a four-choiceolfactometer to untreated (control) cotton plants infested vs. cottonplants treated with PGPR Blend 9 infested, PGPR Blend 9 uninfested, orblank control (empty chamber). Plants were infested with 30 H. virescenscaterpillars. Thirty-two parasitoids were tested each day and replicatedfour times. Means followed by different letters are significantlydifferent (P<0.05, ANOVA, Tukey-Kramer HSD multiple comparison test,n=4)

FIG. 8. Responses of naïve female M. croceipes in a four-choiceolfactometer to untreated (control) cotton plants infested vs. cottonplants treated with PGPR Blend 9 infested, PGPR Blend 9 uninfested, orblank control (empty chamber). Plants were infested with two H.virescens caterpillars. Thirty-two parasitoids were tested each day andreplicated four times. Means followed by different letters aresignificantly different (P<0.05, ANOVA, Tukey-Kramer HSD multiplecomparison test, n=4)

FIG. 9. Effect of PGPR on number of egg batches layed (A); and number oftotal eggs layed (B).

FIG. 10. Chromatographic profiles of headspace volatiles from untreated(control) cotton plants vs. cotton plants treated with PGPR strain MBI600, or PGPR strain ABU 288. Arrows denote volatiles peaks detected inPGPR-treated plants but not in untreated (control) plants.

DETAILED DESCRIPTION

The disclosed subject matter is further described below.

Unless otherwise specified or indicated by context, the terms “a”, “an”,and “the” mean “one or more.” For example, “a peptide” should beinterpreted to mean “one or more peptides” unless otherwise specified orindicated by context.

As used herein, “about”, “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of the term which are not clear to persons of ordinaryskill in the art given the context in which it is used, “about” and“approximately” will mean plus or minus ≦10% of the particular term and“substantially” and “significantly” will mean plus or minus >10% of theparticular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising.”

The term “plant” as utilized herein should be interpreted broadly andmay include angiosperms and gymnosperms, dicots and monocots, and trees.Examples of angiosperm dicots may include, but are not limited totomato, tobacco, cotton, rapeseed, field beans, soybeans, peppers,lettuce, peas, alfalfa, clover, cabbage, broccoli, cauliflower, brusselsprouts), radish, carrot, beets, eggplant, spinach, cucumber, squash,melons, cantaloupe, and sunflowers. Example of angiosperm monocots mayinclude, but are not limited to asparagus, field and sweet corn, barley,wheat, rice, sorghum, onion, pearl millet, rye, oats, and sugar cane.Woody plants may include, but are not limited to fruit trees, acacia,alder, aspen, beech, birch, sweet gum, sycamore, poplar, willow, fir,pine, spruce, larch, cedar, and hemlock.

The term “plant growth promoting rhizobacteria” or “PGPR” refers to agroup of bacteria that colonize plant roots, and in doing so, promoteplant growth and/or reduce disease or damage from predators. Bacteriathat are PGPR may belong to genera including, but not limited toActinobacter, Alcaligenes, Bacillus, Burkholderia, Buttiauxella,Enterobacter, Klebsiella, Kluyvera, Pseudomonas, Rahnella, Ralstonia,Rhizobium, Serratia, Stenotrophomonas, Paenibacillus, andLysinibacillus.

The term “volatile organic compound” or “VOC” refers to an organiccompound that normally is gaseous under ambient conditions. As usedherein, VOCs may include, but are not limited to of alpha-pinene,beta-pinene, beta-myrcene, cis-3-hexenyl acetate, limonene,beta-ocimene, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene, methylsalicylate, decanal, cis-jasmone, caryophyllene, alpha-humulene,beta-farnesene, and mixtures thereof. As disclosed herein, PGPR havebeen identified which induce plants to emit VOCs.

The presently disclosed PGPR may be formulated as an inoculant for aplant. The term “inoculant” means a preparation that includes anisolated culture of a PGPR and optionally a carrier, which may include abiologically acceptable medium.

The presently disclosed PGPR may be isolated or substantially purified.The terms “isolated” or “substantially purified” refers to PGPR thathave been removed from a natural environment and have been isolated orseparated, and are at least 60% free, preferably at least 75% free, andmore preferably at least 90% free, even more preferably at least 95%free, and most preferably at least 100% free from other components withwhich they were naturally associated. An “isolated culture” refers to aculture of the PGPR that does not include significant amounts of othermaterials such as other materials which normally are found in soil inwhich the PGPR grows and/or from which the PGPR normally may beobtained. An “isolated culture” may be a culture that does not includeany other biological, microorganism, and/or bacterial species inquantities sufficient to interfere with the replication of the “isolatedculture.” Isolated cultures of PGPR may be combined to prepare a mixedculture of PGPR.

The genus Bacillus as used herein refers to a genus of Gram-positive,rod-shaped bacteria which are members of the division Firmicutes. Understressful environmental conditions, the Bacillus bacteria produce ovalendospores that can stay dormant for extended periods. Bacillus bacteriamay be characterized and identified based on the nucleotide sequence oftheir 16S rRNA or a fragment thereof (e.g., approximately a 1000 nt,1100 nt, 1200 nt, 1300 nt, 1400 nt, or 1500 nt fragment of 16S rRNA orrDNA nucleotide sequence). Bacillus bacteria may include, but are notlimited to B. acidiceler, B. acidicola, B. acidiproducens, B. aeolius,B. aerius, B. aerophilus, B. agaradhaerens, B. aidingensis, B. akibai,B. alcalophilus, B. algicola, B. alkalinitrilicus, B. alkalisediminis,B. alkalitelluris, B. altitudinis, B. alveayuensis, B.amyloliquefaciens, B. anthracis, B. aquimaris, B. arsenicus, B.aryabhattai, B. asahii, B. atrophaeus, B. aurantiacus, B. azotoformans,B. badius, B. barbaricus, B. bataviensis, B. beijingensis, B.benzoevorans, B. beveridgei, B. bogoriensis, B. boroniphilus, B.butanolivorans, B. canaveralius, B. carboniphilus, B. cecembensis, B.cellulosilyticus, B. cereus, B. chagannorensis, B. chungangensis, B.cibi, B. circulans, B. clarkii, B. clausii, B. coagulans, B.coahuilensis, B. cohnii, B. decisifrondis, B. decolorationis, B.drentensis, B. farraginis, B. fastidiosus, B. firmus, B. flexus, B.foraminis, B. fordii, B. foris, B. fumarioli, B. funiculus, B.galactosidilyticus, B. galliciensis, B. gelatini, B. gibsonii, B.ginsengi, B. ginsengihumi, B. graminis, B. halmapalus, B. halochares, B.halodurans, B. hemicellulosilyticus, B. herbertsteinensis, B. horikoshi,B. horneckiae, B. horti, B. humi, B. hwajinpoensis, B. idriensis, B.indicus, B. infantis, B. infernus, B. isabeliae, B. isronensis, B.jeotgali, B. koreensis, B. korlensis, B. kribbensis, B. krulwichiae, B.lehensis, B. lentus, B. licheniformis, B. litoralis, B. locisalis, B.luciferensis, B. luteolus, B. macauensis, B. macyae, B. mannanilyticus,B. mariflavi, B. marmarensis, B. massiliensis, B. megaterium, B.methanolicus, B. methylotrophicus, B. mojavensis, B. muralis, B.murimartini, B. mycoides, B. nanhaiensvis, B. nanhaiisediminis, B.nealsonii, B. neizhouensis, B. niabensis, B. niacini, B. novalis, B.oceanisediminis, B. odysseyi, B. okhensis, B. okuhidensis, B. oleronius,B. oshimensis, B. panaciterrae, B. patagoniensis, B. persepolensis, B.plakortidis, B. pocheonensis, B. polygoni, B. pseudoalcaliphilus, B.pseudofirmus, B. pseudomycoides, B. psychrosaccharolyticus, B. pumilus,B. qingdaonensis, B. rigui, B. ruris, B. safensis, B. salarius, B.saliphilus, B. schlegelii, B. selenatarsenatis, B. selenitireducens, B.seohaeanensis, B. shackletonii, B. siamensis, B. simplex, B. siralis, B.smithii, B. soli, B. solisalsi, B. sonorensis, B. sporothermodurans, B.stratosphericus, B. subterraneus, B. subtilis, B. taeansis, B.tequilensis, B. thermantarcticus, B. thermoamylovorans, B.thermocloacae, B. thermolactis, B. thioparans, B. thuringiensis, B.tripoxylicola, B. tusciae, B. vallismortis, B. vedderi, B. vietnamensis,B. vireti, B. wakoensis, B. weihenstephanensis, B. xiaoxiensis, andmixtures or blends thereof.

The PGPR and inoculants thereof disclosed herein may include B.amyloliquefaciens or a Bacillus species that is closely related to B.amyloliquefaciens. The partial sequence of B. amyloliquefaciens strainChilli-1 16S ribosomal rDNA (GenBank Accession No. HQ021420.1) isprovided herein as SEQ ID NO:1. A Bacillus species that is closelyrelated to B. amyloliquefaciens may be defined as a species having a 16SrDNA sequence comprising SEQ ID NO:1 or comprising a 16S rDNA sequencehaving at least about 98% or 99% sequence identity to SEQ ID NO:1.

The PGPR and inoculants thereof disclosed herein may include B.mojavensis or a Bacillus species that is closely related to B.mojavensis. The partial sequence of B. mojavensis strain NBSL51 16Sribosomal rDNA (GenBank Accession No. JN624928.1) is provided herein asSEQ ID NO:2. A Bacillus species that is closely related to B. mojavensismay be defined as a species having a 16S rDNA sequence comprising SEQ IDNO:2 or comprising a 16S rDNA sequence having at least about 98% or 99%sequence identity to SEQ ID NO:2.

The PGPR and inoculants thereof disclosed herein may include B.solisalsi or a Bacillus species that is closely related to B. solisalsi.The partial sequence of B. solisalsi strain YC1 16S ribosomal rDNA(GenBank Accession No. NR_044387) is provided herein as SEQ ID NO:3. ABacillus species that is closely related to B. solisalsi may be definedas a species having a 16S rDNA sequence comprising SEQ ID NO:3 orcomprising a 16S rDNA sequence having at least about 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3.

The PGPR and inoculants thereof disclosed herein may include B. pumilusor a Bacillus species that is closely related to B. pumilus. The partialsequence of B. pumilus strain TUB1 16S ribosomal rDNA (GenBank AccessionNo. HE613653.1) is provided herein as SEQ ID NO:4. A Bacillus speciesthat is closely related to B. pumilus may be defined as a species havinga 16S rDNA sequence comprising SEQ ID NO:4 or comprising a 16S rDNAsequence having at least about 96%, 97%, 98%, or 99% sequence identityto SEQ ID NO:4.

The PGPR and inoculants thereof disclosed herein may include B. simplexor a Bacillus species that is closely related to B. simplex. The partialsequence of B. simplex strain NH.259 16S ribosomal rDNA (GenBankAccession No. EU627171.1) is provided herein as SEQ ID NO:5. A Bacillusspecies that is closely related to B. simplex may be defined as aspecies having a 16S rDNA sequence comprising SEQ ID NO:5 or comprisinga 16S rDNA sequence having at least about 93%, 94%, 95%, 96%, 97%, 98%,or 99% sequence identity to SEQ ID NO:5.

The PGPR and inoculants thereof disclosed herein may include B. subtilisor a Bacillus species that is closely related to B. subtilis. Thepartial sequence of B. subtilis strain NH.259 16S ribosomal rDNA(GenBank Accession No. EU627171.1) is provided herein as SEQ ID NO:6. ABacillus species that is closely related to B. subtilis may be definedas a species having a 16S rDNA sequence comprising SEQ ID NO:5 orcomprising a 16S rDNA sequence having at least about 98%, or 99%sequence identity to SEQ ID NO:6.

In some embodiments of the inoculants disclosed herein comprisingBacillus bacteria, the Bacillus species is not B. subtilis and is not aBacillus species that is closely related to B. subtilis. A Bacillusspecies that is not closely related to B. subtilis may be defined as aspecies having a 16S rDNA sequence that has no more than 99%, 98%, 97%,96%, 95%, 94%, 93%, 92%, or 91% sequence identity to SEQ ID NO:5.

“Percentage sequence identity” may be determined by aligning twosequences of equivalent length using the Basic Local Alignment SearchTool (BLAST) available at the National Center for BiotechnologyInformation (NCBI) website (i.e., “bl2seq” as described in Tatiana A.Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool forcomparing protein and nucleotide sequences”, FEMS Microbiol Lett.174:247-250, incorporated herein by reference in its entirety). Forexample, percentage sequence identity between SEQ ID NO:1 and SEQ IDNO:5 may be determined by aligning these two sequences using the onlineBLAST software provided at the NCBI website.

“Percentage sequence identity” between two deoxyribonucleotide sequencesmay also be determined using the Kimura 2-parameter distance model whichcorrects for multiple hits, taking into account transitional andtransversional substitution rates, while assuming that the fournucleotide frequencies are the same and that rates of substitution donot vary among sites (Nei and Kumar, 2000) as implemented in the MEGA 4(Tamura K, Dudley J, Nei M & Kumar S (2007) MEGA4: MolecularEvolutionary Genetics Analysis (MEGA) software version 4.0. MolecularBiology and Evolution 24:1596-1599), preferably version 4.0.2 or later.The gap opening and extension penalties are set to 15 and 6.66respectively. Terminal gaps are not penalized. The delay divergentsequences switch is set to 30. The transition weight score is 35 set to0.5, as a balance between a complete mismatch and a matched pair score.The DNA weight matrix used is the IUB scoring matrix where x's and n'sare matches to any IUB ambiguity symbol, and all matches score 1.9, andall mismatched score O.

As used herein, “Blend 8” refers to a mixture of Bacillus bacteriaincluding Bacillus amyloliquefaciens strain AP-188, Bacillus mojavensisstrain AP-209, Bacillus solisalsi strain AP-217, and Bacillusamyloliquefaciens strain AP-218. (See Table 1). As used herein, “Blend9” refers to a mixture of Bacillus bacteria including Bacillusamyloliquefaciens strain AP-136, Bacillus mojavensis strain AP-188,Bacillus solisalsi strain AP-219, and Bacillus amyloliquefaciens strainAP-295.

The presently disclosed PGPR may be utilized to treat plants and induceVOC production in the treated plants. For example, the presentlydisclosed PGPR may be formulated as an inoculant for treating plants.The methods of treatment contemplated herein may include treating aplant directly including treating leaves, stems, or roots of the plantdirectly. The methods of treatment contemplated herein may includetreating seeds of the plant, e.g., prior to the seeds being planted toproduce a treated plant. The methods contemplated herein also mayinclude treating a plant indirectly, for example, by treating soil orthe environment surrounding the plant (e.g., in-furrow granular orliquid applications). Suitable methods of treatment may include applyingan inoculant including the PGPR via high or low pressure spraying,drenching, and/or injection. Plant seeds may be treated by applying lowor high pressure spraying, coating, immersion, and/or injection. Afterplant seeds have been thusly treated, the seeds may be planted andcultivated to produce plants. Plants propagated from such seeds may befurther treated with one or more applications. Suitable applicationconcentrations may be determined empirically. In some embodiments wherethe PGPR are applied as a spray to plants, suitable applicationconcentrations may include spraying 10⁶-10¹⁸ colony forming units (cfu)per hectare of plants, more commonly 10⁷-10¹⁵ cfu per hectare. Forcoated seeds, in some embodiments, suitable application concentrationsmay be between 10²-10⁸ cfu per seed, preferably 10⁴-10⁷ cfu per seed. Inother embodiments, the PGPR may be applied as a seedling root-dip or asa soil drench at a concentration of about 10²-10¹² cfu/ml, 10⁴-10¹⁰cfu/ml, or about 10⁶-10⁸ cfu/ml.

The PGPR may be applied together with a suitable carrier in acomposition (e.g., such as an inoculum). Suitable carriers may include,but are not limited to, water or other aqueous solutions, slurries,solids (e.g., peat, wheat, bran, vermiculite, and pasteurized soil) ordry powders. In some embodiments, the composition includes 10²-10¹² cfuper ml carrier, or 10⁴-10¹⁰ cfu per ml carrier, or 10⁶-10⁸ cfu per mlcarrier. The composition may include additional additives includingbuffering agents, surfactants, adjuvants, or coating agents.

The presently disclosed methods may be performed in order to modifyinsect behavior towards a treated plant. As used herein, “modifying”insect behavior may include reducing or preventing negative insectbehavior and/or increasing positive insect behavior. Reducing orpreventing negative insect behavior may include reducing or preventingdamage from insects. For example, the methods may be practiced to reduceor prevent feeding of herbivorous insects on a treated plant.Preferably, the methods reduce feeding on a treated plant versus anuntreated plant by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90%. Reduction in feeding may be measured by comparing body massof larvae feeding on treated plants versus untreated plants over aperiod of time. Reduction in feeding also may be measured by comparingmass of the plant lost due to insect feeding per time. The methods alsomay be practiced to reduce or prevent egg-laying of herbivorous insectson a treated plant. Preferably, the methods reduce egg-laying (i.e.,oviposition) on a treated plant versus an untreated plant by at leastabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. Reduction inegg-laying may be measured by comparing egg-laying per insect (e.g.,total number of eggs and/or total number of egg batches) on a treatedplant versus an untreated plant. Herbivorous insects whose behavior maybe modified by the presently disclosed methods may include, but are notlimited to, Spodoptera exigua and Pieris rapae. The methods also may bepracticed to attract natural enemies of insects to treated plants,including but not limited to predatory insects or insect parasitoids.Predatory insects may include, but are not limited to, lady beetles(i.e., Coccinelidae, assassin bugs (i.e., Reduviidae), big-Eyed bugs(i.e., Geocoridae), minute pirate bug (i.e., Antrocoridae), damsel bug(i.e., Nabidae), lacewings (i.e., Neuroptera), and predatory mites(i.e., Phytoseiidae). Insect parasitoids may include, but are notlimited to, Brachonid wasps (e.g. Cotesia marginiventris, Microplitiscroceipes, Cotesia rubecula, and Aphidius colemani), Ichneumonid wasps,Chalcid wasps (e.g., Eretmocerus spp., and Encarsia formosa), andTachinid flies.

ILLUSTRATIVE EMBODIMENTS

The following embodiments are illustrative and are not intended to limitthe claimed subject matter.

Embodiment 1

Isolated plant growth producing rhizobacteria (PGPR) that induceproduction of one or more volatile organic compounds (VOCs) by a plantthat has been treated with the PGPR, and optionally the PGPR areselected from genus selected from a group consisting of Actinobacter,Alcaligenes, Bacillus, Burkholderia, Buttiauxella, Enterobacter,Klebsiella, Kluyvera, Pseudomonas, Rahnella, Ralstonia, Rhizobium,Serratia, Stenotrophomonas, Paenibacillus, and Lysinibacillus.

Embodiment 2

The PGPR according to embodiment 1 or 2, wherein the one or more VOCscomprise one or more compounds selected from a group consisting of ThePGPR and inoculants thereof.

Embodiment 3

The PGPR according to any of the preceding embodiments, wherein the oneor more VOCs modify behavior of an insect exposed to the one or moreVOCs.

Embodiment 4

The PGPR according to any of the preceding embodiments, wherein theinsect is an herbivore and the one or more VOCs reduce egg-laying of theinsect on the plant.

Embodiment 5

The PGPR according to any of the preceding embodiments, wherein theinsect is an herbivore and the one or more VOCs reduce feeding of theinsect on the plant.

Embodiment 6

The PGPR according to any of the preceding embodiments, wherein theinsect is a predator or a parasitoid and the one or more VOCs attractthe predator or the parasitoid to the plant.

Embodiment 7

The PGPR according to any of the preceding embodiments, wherein the PGPRare Bacillus bacteria selected from a group consisting of B. acidiceler,B. acidicola, B. acidiproducens, B. aeolius, B. aerius, B. aerophilus,B. agaradhaerens, B. aidingensis, B. akibai, B. alcalophilus, B.algicola, B. alkalinitrilicus, B. alkalisediminis, B. alkalitelluris, B.altitudinis, B. alveayuensis, B. amyloliquefaciens, B. anthracis, B.aquimaris, B. arsenicus, B. aryabhattai, B. asahii, B. atrophaeus, B.aurantiacus, B. azotoformans, B. badius, B. barbaricus, B. bataviensis,B. beijingensis, B. benzoevorans, B. beveridgei, B. bogoriensis, B.boroniphilus, B. butanolivorans, B. canaveralius, B. carboniphilus, B.cecembensis, B. cellulosilyticus, B. cereus, B. chagannorensis, B.chungangensis, B. cibi, B. circulans, B. clarkii, B. clausii, B.coagulans, B. coahuilensis, B. cohnii, B. decisifrondis, B.decolorationis, B. drentensis, B. farraginis, B. fastidiosus, B. firmus,B. flexus, B. foraminis, B. fordii, B. fortis, B. fumarioli, B.funiculus, B. galactosidilyticus, B. galliciensis, B. gelatini, B.gibsonii, B. ginsengi, B. ginsengihumi, B. graminis, B. halmapalus, B.halochares, B. halodurans, B. hemicellulosilyticus., B.herbertsteinensis, B. horikoshi, B. horneckiae, B. horti, B. humi, B.hwajinpoensis, B. idriensis, B. indicus, B. infantis, B. infernus, B.isabeliae, B. isronensis, B. jeotgali, B. koreensis, B. korlensis, B.kribbensis, B. krulwichiae, B. lehensis, B. lentus, B. licheniformis, B.litoralis, B. locisalis, B. luciferensis, B. luteolus, B. macauensis, B.macyae, B. mannanilyticus, B. marisflavi, B. marmarensis, B.massiliensis, B. megaterium, B. methanolicus, B. methylotrophicus, B.mojavensis, B. muralis, B. murimartini, B. mycoides, B. nanhaiensis, B.nanhaiisediminis, B. nealsonii, B. neizhouensis, B. niabensis, B.niacini, B. novalis, B. oceanisediminis, B. odysseyi, B. okhensis, B.okuhidensis, B. oleronius, B. oshimensis, B. panaciterrae, B.patagoniensis, B. persepolensis, B. plakortidis, B. pocheonensis, B.polygoni, B. pseudoalcaliphilus, B. pseudofirmus, B. pseudomycoides, B.psychrosaccharolyticus, B. pumilus, B. qingdaonensis, B. rigui, B.ruris, B. safensis, B. salarius, B. saliphilus, B. schlegelii, B.selenatarsenatis, B. selenitireducens, B. seohaeanensis, B.shackletonii, B. siamensis, B. simplex, B. siralis, B. smithii, B. soli,B. solisalsi, B. sonorensis, B. sporothermodurans, B. stratosphericus,B. subterraneus, B. subtilis, B. taeansi, B. tequilensis, B.thermantarcticus, B. thermoamylovorans, B. thermocloacae, B.thermolactis, B. thioparans, B. thuringiensis, B. tripoxylicola, B.tusciae, B. vallismortis, B. vedderi, B. vietnamensis, B. vireti, B.wakoensis, B. weihenstephanensis, B. xiaoxiensis, and mixtures or blendsthereof.

Embodiment 8

The Bacillus bacteria according to embodiment 7, wherein the bacteriahave a 16S rDNA nucleic acid sequence comprising SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

Embodiment 9

The Bacillus bacteria according to embodiment 7, wherein the bacteriahave a 16S rDNA nucleic acid sequence that is at least 98% identical toSEQ ID NO:1.

Embodiment 10

The Bacillus bacteria according to any of embodiments 7-9, wherein thebacteria have a 16S rDNA nucleic acid sequence that is at least 98%identical to SEQ ID NO:2.

Embodiment 11

The Bacillus bacteria according to any of embodiments 7-10, wherein thebacteria have a 16S rDNA nucleic acid sequence that is at least 91%identical to SEQ ID NO:3.

Embodiment 12

The Bacillus bacteria according to any of embodiments 7-11, wherein thebacteria have a 16S rDNA nucleic acid sequence that is at least 96%identical to SEQ ID NO:4.

Embodiment 13

The Bacillus bacteria according to any of embodiments 7-12, wherein thebacteria have a 16S rDNA nucleic acid sequence that is at least 93%identical to SEQ ID NO:5.

Embodiment 14

The Bacillus bacteria according to any of embodiments 7-13, wherein thebacteria have a 16S rDNA nucleic acid sequence that is at least 98%identical to SEQ ID NO:6.

Embodiment 14

The Bacillus bacteria according to embodiment 7, wherein the bacteriaare selected from a group consisting of Bacillus amyloliquefaciens,Bacillus mojavensis, Bacillus solisalsi, Bacillus pumilus, Bacillussimplex, Bacillus subtilis and mixtures thereof.

Embodiment 15

The Bacillus bacteria according to embodiment 7, wherein the bacteriaare Bacillus amyloliquefaciens.

Embodiment 16

The Bacillus bacteria according to embodiment 15, wherein the bacteriaare selected from a group consisting of Bacillus amyloliquefaciensstrain AP-136, Bacillus amyloliquefaciens strain AP-188, Bacillusamyloliquefaciens strain AP-218, Bacillus amyloliquefaciens strainAP-219, and Bacillus amyloliquefaciens strain AP-295.

Embodiment 17

The Bacillus bacteria according to embodiment 7, wherein the bacteriaare Bacillus mojavensis.

Embodiment 18

The Bacillus bacteria according to embodiment 17, wherein the bacteriaare Bacillus mojavensis strain AP-209.

Embodiment 19

The Bacillus bacteria according to embodiment 7, wherein the bacteriaare Bacillus solisalsi.

Embodiment 20

The Bacillus bacteria according to embodiment 19, wherein the bacteriaare Bacillus solisalsi strain AP-217.

Embodiment 21

The Bacillus bacteria according to embodiment 7, wherein the bacteriaare Bacillus pumilus.

Embodiment 22

The Bacillus bacteria according to embodiment 21, wherein the bacteriaare Bacillus pumilus strain INR7.

Embodiment 23

The Bacillus bacteria according to embodiment 7, wherein the bacteriaare Bacillus simplex.

Embodiment 24

The Bacillus bacteria according to embodiment 23, wherein the bacteriaare Bacillus simplex strain ABU 288.

Embodiment 25

The Bacillus bacteria according to embodiment 7, wherein the bacteriaare Bacillus subtilis strain MBI 600.

Embodiment 26

The Bacillus bacteria according to embodiment 7, wherein the bacteriacomprise a mixture of Bacillus species.

Embodiment 27

An inoculant for a plant comprising the PGPR of any of the precedingembodiments and a carrier.

Embodiment 28

The inoculant of embodiment 27 further comprising a phytohormone, ananti-microbial compound, or both.

Embodiment 29

The inoculant of embodiment 28, wherein the phytohormone is selectedfrom a group consisting of acetoin, 2,3-butanediol, and indole-aceticacid and the anti-microbial compound is selected from a group consistingof phenylethanol and 4-hydroxybenzoate.

Embodiment 30

A method of modifying insect behavior towards a plant, the methodcomprising a administering the inoculant of any of embodiments 27-29 tothe plant, to seeds of the plant, or to soil surrounding the plant.

Embodiment 31

The method according to embodiment 30, wherein the insect is anherbivore and the method reduces egg-laying of the insect on the plant.

Embodiment 32

The method according to embodiment 30 or 31, wherein the insect is anherbivore and the method reduces feeding of the insect on the plant.

Embodiment 33

The method according to any of embodiments 30-32, wherein the insect isa predator or a parasitoid and the method attracts the predator orparasitoid to the plant.

Embodiment 34

The method according to any of embodiments 30-33, wherein the plant isselected from a group consisting of alfalfa, rice, barley, rye, cotton,sunflower, peanut, corn, potato, sweet potato, bean, pea, lentilchicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip,turnip, cauliflower, broccoli, turnip, radish, spinach, onion, garlic,eggplant pepper, celery, carrot, squash, pumpkin, zucchini, cucumber,apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple,soybean, canola, oil seed rape, spring wheat, winter wheat, tobacco,tomato, sorghum, and sugarcane.

EXAMPLES

The following Examples are illustrative and are not intended to limitthe scope of the claimed subject matter.

Example 1 Effects of Plant Growth-Promoting Rhizobacteria on Inductionof Cotton Plant Volatiles and Attraction of Parasitoids

Abstract

Parasitic wasps (parasitoids) are known to utilize as host location cuesvarious types of host-related volatile signals. These volatile signalscould be plant-based, originate from the herbivore host, or be producedfrom an interaction between herbivores and their plant host. The successof parasitoids in suppressing pest populations depends on their abilityto locate hosts in a complex olfactory and visual environment. Despitethe intense interest in host-parasitoid interactions, certain aspects ofolfactory communication in this group of insects are not wellunderstood.

Here, studies were conducted to evaluate the potential of plantgrowth-promoting rhizobacteria (PGPR) on the induction of cottonvolatiles and consequences for response of parasitoids. Three PGPRtreatments were evaluated: i) Bacillus pumilis strain INR-7 and twoblends of Bacillus bacteria. An untreated (water) control was alsotested. There were quantitative and qualitative differences in headspacevolatiles collected from PGPR-treated and untreated cotton plants. Atotal of eleven peaks representing VOCs were detected from headspace ofPGPR-treated cotton plants but only three peaks were detected inuntreated cotton plants. Differences in root growth between PGPR-treatedvs. untreated plants were recorded.

Introduction

Plant Growth-Promoting Rhizobacteria (PGPR) represent a wide range ofroot-colonizing bacteria whose application is often associated withincreased rates of plant growth (Kloepper 1992, Zehnder et al. 1997,Kloepper et al. 2004), suppression of soil pathogens (Schippers et al.1987, Burkett-Cadena et al. 2008), and the induction of systemicresistance against insect pests (van Loon et al. 1998, Kloepper et al.1999, Ramamoorthy et al. 2001, Zehnder et al. 2001, Ryu et al. 2004, Jiet al. 2006). PGPR-based inoculants include formulations containing asingle strain, a mixture of two strains, or complex mixtures of Bacillusspp. (Lucy et al. 2004, Kloepper and Ryu, 2006). The effects ofapplication of PGPR on induction of volatile organic compounds (VOCs) intreated plants are virtually unexamined, despite evidence that inductionof plant volatiles is dependent on many factors. The interactionsioticfactors which include plant hormones (de Bruxelles and Roberts, 2001,Thaler et al. 2002, Farmer et al. 2003, Ament et al. 2004),herbivore-derived elicitors (Mattiaci et al. 1995, Albom et al. 1997,Spiteller and Boland, 2003), and associated microorganisms includingpathogens (Preston et al. 1999, Cardoza et al. 2002), as well as abioticfactors including wounding (Mithöfer et al. 2005), heavy metals(Mithöfer et al. 2004) and temperature and light (Takabayashi et al.1994, Gouinguene and Turlings 2002). The lack of research related to theeffects of PGPR on induction of plant volatiles is surprising given thatPGPR are increasingly being applied to production of several field cropsincluding cotton (Gossypium hirsutum L.), tomato (Solanum lycopersicumL.), watermelon (Citrullus lanatus Thunb.), and pearl millet (Pennisetumglaucum) in the USA or India (Glick 1995, Backman et al. 1997,Cleyet-Marcel et al. 2001, Kokalis-Burelle et al. 2003, Niranjan Raj etal. 2003, Burkett-Cadena et al. 2008). In 1997, Backman et al. reportedthat 60-75% of the US cotton crop was being treated with the PGPRproduct Kodiak®, a Bacillus subtilis product used for suppression ofFusarium and Rhizoctonia soil pathogens. PGPR have previously been usedto treat agricultural crops on a large scale.

Like herbivores that use VOCs in their search for suitable host plants(Dicke et al. 2000), parasitic insects are also known to use blends ofVOCs for foraging and host location of their herbivore hosts (Turlingset al. 1990, McCall et al. 1993, De Moraes et al. 1998). These VOCs canoriginate from the plant, herbivore host, or be the result of aninteraction between herbivores and the plant (McCall et al., 1994,Cortesero et al. 1997). Plant-based VOCs are further categorized intogreen leaf volatiles (GLVs), which are released immediately in responseto mechanical damage or at the beginning of herbivore feeding, andherbivore-induced plant volatiles (HIPVs), which are emitted as adelayed response to herbivore feeding damage. These blends of VOCs,which are highly attractive to parasitoids of cotton herbivoresincluding Microplitis croceipes (Cresson) and Cotesia marginiventris(Cresson) (Hymenoptera: Braconidae), are released in response tocaterpillar feeding (De Moraes et al. 1998, Chen and Fadamiro 2007,Ngumbi et al. 2009, 2010). It is possible that PGPR could affect VOCproduction in cotton with important consequences for foragingparasitoids and other chemically mediated insect-plant and tri-trophicinteractions.

Here, the hypothesis that PGPR could elicit changes in cotton plant VOCsand alter the growth of cotton roots was tested. Additionally, it washypothesized that parasitoids of cotton herbivores would show greaterattraction to PGPR-treated cotton plants compared to untreated cottonplants via changes in the emission of VOCs. PGPR-treated and untreatedcotton plants were grown under greenhouse conditions and headspacevolatiles collected 4-6 weeks post planting. Coupled gaschromatography-mass spectrometry (GC-MS) was used to identify andanalyze headspace volatiles from PGPR-treated and untreated cottonplants. A four-choice olfactometer was used to study the behavior of M.croceipes when presented with PGPR-treated plants versus untreatedplants. To the inventors' knowledge, this is the first report of PGPRaffecting the production of VOCs by cotton plants.

Materials and Methods

PGPR Strains.

As shown in Table 1 a total of eight strains of Bacillus spp. (all fromAuburn University) were used to develop the three PGPR treatmentsstudied: i) Bacillus pumilus strain INR-7 (AP 18), ii) Blend 8,containing four strains of Bacillus spp. (AP 188, 209, 217 218), andiii) Blend 9, containing four strains of Bacillus spp. (AP 136, 188,219, 295).

TABLE 1 PGPR Preparations PGPR Preparation Identification Blend 8Bacillus amyloliquefaciens strain AP-188 Bacillus mojavensis strainAP-209 Bacillus solisalsi strain AP-217 Bacillus amyloliquefaciensstrain AP-218 Blend 9 Bacillus amyloliquefaciens strain AP-136 Bacillusamyloliquefaciens strain AP-188 Bacillus amyloliquefaciens strain AP-219Bacillus amyloliquefaciens strain AP-295 INR-7 Bacillus pumilus strainAP-18

Plants.

Conventional variety (G. hirsutum) Max-9 cotton seeds (All-Tex Seed,Inc.) were grown individually in round plastic pots (9 cm high, 11 cmdiameter) filled with a top soil/vermiculite/peat moss mixture. Theseeds were then grown in individual pots (9 cm high, 11 cm diameter) ina greenhouse (Auburn University Plant Science Greenhouse Facility) at25° C.±10, 15:9 h (L/D) photoperiod, and 50±10% relative humidity. PGPRtreatments were applied at seeding (1 ml/seed) as aqueous sporesuspensions (1×10⁷ spores/ml). Weekly, PGPR-treated plants received 1 mladditional treatments as an aqueous bacterial suspension (1×10⁹ cfu/ml).Plants used for headspace volatile collections were 4 to 6 weeks oldfrom day of planting.

Insects.

Parent cultures of M. croceipes were provided by the USDA-ARS, InsectBiology and Population Management Research Laboratory (Tifton, Ga.).Microplitis croceipes were reared on caterpillars of Heliothis virescens(Fab.) Lepidoptera: Noctuidae, its preferred host (Stadelbacher et al.1984, King et al. 1985), using a procedure similar to that of Lewis andBurton (1970). Eggs purchased from Benzone Research (Carlisle, Pa., USA)were used to start a laboratory colony of H. virescens reared on alaboratory-prepared pinto bean diet (Shorey and Hale 1965). All colonieswere maintained at 25±1° C., 75±5% RH, and under a L14:D10 photoperiod.Newly emerged M. croceipes adults were collected prior to mating, sexed,and placed in pairs of individuals of opposite sex (mated individuals)in a 6-cm diameter plastic Petri dish supplied with water and sugarsources. Water was provided by filling a 0.5 ml microcentrifuge tubewith distilled water and threading a cotton string through a hole in thecap of the tube. About 5 drops (2 μl per drop) of 10% sugar solutionwere smeared on the inside of the Petri dish cover with a cotton-tippedapplicator. Naïve parasitoids (aged 3-5 days) were used for thebioassays.

Collection and GC Analysis of Headspace Volatiles.

The methodology and protocols used for volatile collection were similarto those reported by Gouinguené et al. (2005), but with somemodifications. Headspace volatiles were collected both from PGPR-treatedand untreated cotton plants as well as PGPR-treated and untreatedcaterpillar damaged cotton plants. In order to detect herbivore inducedplant volatiles (HIPVs) from PGPR-treated and untreated plants, 302^(nd) instar caterpillars of Heliothis virescens Fab. (Lepidoptera:Noctuidae) were allowed to feed on a potted cotton plant for 12 h priorto volatile collection. The pot with the potting soil was wrapped withaluminum foil to minimize evaporation of water and volatiles from thesoil. The plant was then placed in a volatile collection chamber(Analytical Research Systems, Inc., Gainesville, Fla.) consisting of a 5L glass jar. A purified (using activated charcoal) air stream of 500ml/min was passed through the jar at room temperature for 24 hr.Headspace volatiles were collected using a trap containing 50 mg ofSuper-Q (Alltech Associates, Deerfield, Ill.) and eluted with 200 μl ofmethylene chloride. The elutions (200 μl) were stored in a freezer (at−20° C.) until use. Another container with potting soil but no plant wasused to check for miscellaneous impurities and background noise. One μlof each headspace volatile extract was injected into a Shimadzu GC-17Aequipped with a flame ionization detector (FID). The dimension of thecapillary column used was as follows: Rtx-1MS, 0.25 mm I.D., 0.25 μmfilm thickness (Restek, Bellefonte, Pa.). Helium was used as carrier gasat a flow rate of 1 ml/min. The GC oven was programmed as follows:inject at 40° C., hold at 40° C. for 2 minutes, and then increase by 5°C./min to 200° C. for a total of 40 minutes. The temperatures of boththe injector and detector were set at 200° C. Five replicates werecarried out.

GC-MS Analysis.

GC profiles of each plant treatment were later identified by GC-MS usingan Agilent 7890A GC coupled to a 5975C Mass Selective Detector, with aHP-5 ms capillary column (30 m×0.25 mm I.D., 0.25 μm film thickness).One pi of each headspace extract was injected into the GC in splitlessmode, using the GC conditions described above. Mass spectra wereobtained using electron impact (EI, 70 eV). Identification of peaks wasdone using NIST 98 library (National Institute of Standards andTechnology, Gaithersburg, Md.) and comparison with published GC profilesof cotton head space volatiles (Thompson et al. 1971, Loughrin et al.1994, McCall et al. 1994). The structures of the identified compoundswere confirmed using commercially available synthetic standards withpurity>97% (as indicated on the labels) obtained from Sigma® ChemicalCo. (St. Louis, Mo.).

Analysis of Cotton Root Growth.

A Separate Experiment was Carried Out to determine if treatment ofcotton with PGPR would lead to differences in cotton root growth. One mlof PGPR (INR-7, Blend 8, and Blend 9) at spore concentrations of 10⁷ wasapplied to cotton seed. The PGPR-treated seeds were then grown inindividual pots (15 cm high, 21 cm diameter) in a greenhouse (AuburnUniversity Plant Science Greenhouse Facility) at 25° C.±10, 15:9 h (L/D)photoperiod, and 50±10% relative humidity. Seeds were planted in a topsoil/vermiculite/peat moss mixture. Additionally, every week, 1 ml ofaqueous bacterial suspension (10⁹) colony forming units (cfu/ml) wasapplied. Plants used for cotton root growth analysis were two weeks old.After washing roots, an analysis of root architecture was made on eachplant's rooting system using the system of Regent Instruments, Inc.(Sainte-Foy, Quebec), which consists of scanner model LA 1600+ andWinRhizo software (version 2004a). Data from the resulting analyses werecollected for two root parameters: root surface area and root volume(0-0.5 and 0.5-1.0 mm). Data on root dry weight were also collected.Eight replicates were done.

Four-Choice Olfactometer Bioassays with Parasitoids.

Attraction of M. croceipes to odors of PGPR-treated vs. untreatedplants, as well as PGPR-treated caterpillar-damaged vs. undamagedplants, was assessed in four-choice olfactometer bioassays (AnalyticalResearch Systems, Gainesville, Fla.). The apparatus was similar to thesystem described by Pettersson (1970) and Kalule and Wright (2004). Itconsists of a central chamber with orifices at the four corners throughwhich purified and humidified air was drawn in, creating four potentialodor fields, and a central orifice where mixing of the airflow from thearms occurred. A constant airflow of 500 ml/min was maintained througheach of the four orifices at the corners of the olfactometer. Mixturesof air from the control arms and volatile odors from the treatment armswere drawn out from the olfactometer, through the central orifice, witha constant airflow of 2.5 l/min. Volatile odors emanated from plantsthat were 4-6 weeks old post-planting. The pot with the potting soil waswrapped with aluminum foil to minimize evaporation of water andvolatiles. The plants were then placed in 5 L glass jar (32 cm high,14.5 cm diameter) volatile collection chambers (Analytical ResearchSystems, Inc., Gainesville, Fla. USA) and purified air (500 ml/min) waspassed through the chambers and into each of the 4 orifices at thecorners of the olfactometer.

Naïve three-to-five-day-old female M. croceipes were used in allexperiments. A wasp was removed from the cage with an aspirator andintroduced singly into a glass tube (1.5 cm). The glass tube wasconnected to the central orifice of the olfactometer to expose the waspto the volatile odors/air mixtures. Once in the chamber, a parasitoidwas given 15 min to make a choice among the four air fields. If theparasitoid had not made a choice within this duration, it was removed,discarded, and not included in the analyses. In order to remove anydirectional bias in the chamber, the olfactometer and the position ofplants were rotated after eight parasitoids had been tested. A total of32 parasitoids were tested each day (8 parasitoids per rotation). Threesets of four-choice olfactometer experiments were conducted to testwhether females M. croceipes responded differently to uninfested versusinfested cotton plants and PGPR-treated versus untreated cotton plants.In the first experiment the following two treatments and two controlswere compared: (1) PGPR INR7-treated plant (2) PGPR Blend 9 treatedplant (3) Untreated (control) plant, (4) blank control (empty chamber).Based on the results of the first experiment, which showed significantattraction of the parasitoid to PGPR Blend 9-treated plants as comparedto untreated (control) plants (FIG. 6), a second experiment wasconducted to determine if PGPR treatment is as effective as caterpillarinfestation/damage in attracting parasitoids to plants. For thisexperiment, the PGPR treatment (Blend 9) was selected based on resultsfrom the previous experiment, and volatile odors from the following werecompared: (1) PGPR Blend 9-treated plant infested, (2) PGPR Blend9-treated plant uninfested, (3) Untreated (control) plant infested, and(4) control (empty chamber). Each plant was infested with 30 H.virescens caterpillars. A third experiment was conducted based on theresult of the second experiment, which showed that untreated (control)plants infested with 30 caterpillars were as attractive to parasitoidsas PGPR Blend 9-treated plants infested with 30 caterpillars. Thissuggests that PGPR treatment may be signaling a lower level ofcaterpillar damage than the level tested in the second experiment. Totest this hypothesis and determine if PGPR treatment is as good as lowlevel of caterpillar damage in attracting parasitoids to plants, thesame treatments and controls tested in experiment 2 were compared buteach infested plant was infested with two H. virescens caterpillars.

Four-choice olfactometer bioassays were carried out between 10:00 and18:00 hrs each day at 25±1° C., 60±5% r.h. and 250 lux. The firstexperiment was replicated five times, while experiments 2 and 3 werereplicated four times. For each replicate, a new set of plants was used.

Statistical Analysis.

Data met the key assumptions of Analysis of Variance and thus were nottransformed prior to analysis. Significant differences in the amounts ofeach volatile component emitted by PGPR-treated (Bacillus pumilis strainINR-7, Blend 8, and Blend 9) treated and untreated plants wereestablished using Analysis of Variance (ANOVA) followed by theTukey-Kramer HSD multiple comparison test (P<0.05, JMP 7.0.1, SASInstitute 2007). Significant differences in cotton root growth wereestablished by ANOVA followed by the Tukey-Kramer HSD multiplecomparison test (P<0.05, JMP 7.0.1, SAS Institute 2007). Four-choiceolfactometer data were analyzed by one-way ANOVA followed by theTukey-Kramer HSD multiple comparison test (P<0.05, JMP 7.0.1, SASInstitute 2007).

Results

GC and GC-MS Analyses of Headspace Volatiles.

The GC profiles of the extracts of headspace volatiles from PGPR-treatedand untreated cotton plants are shown in FIG. 1. A total of 11 peaks(volatile components) were detected in the headspace of PGPR-treated(INR-7, Blend 8, and Blend 9) cotton plants (FIG. 1). These peaks, asidentified by GC-MS, included α-pinene, β-pinene, β-myrcene,cis-3-hexenyl acetate, limonene, (β)-ocimene, linalool, caryophyllene,α-humulene, and β-farnesene. Most of these peaks were not detected orwere detected in insignificant amounts in the headspace of untreatedcotton plants (FIG. 1). Only three peaks (components) were detectable inuntreated cotton plants and were identified by GC-MS as α-pinene,cis-3-hexenyl acetate, and caryophyllene. However, all three componentswere detected in much greater amounts in the headspace of PGPR-treatedplants. Additionally, significant differences were recorded between thePGPR treatments. (See Table 2).

TABLE 2 Composition of headspace volatiles emitted by untreated(control) cotton plants vs. cotton plants treated with strain INR-7,Blend 8, or Blend 9 Cotton plants Cotton plants Untreated Cotton plantstreated with treated with (control) cotton treated with PGPR PGPR IDCompound^(a) plants PGPR strain INR-7 Blend 8 Blend 9 1 α-pinene 58 ±12^(d) 12,960 ± 2288^(a) 9,766 ± 1011^(b) 5,714 ± 519^(c) 2 β-pinene0^(d)  2,739 ± 1782^(a) 2,298 ± 280^(b)   786 ± 132^(c) 3 β-myrcene0^(d) 4,084 ± 105^(a) 3,044 ± 94^(b)   864 ± 148^(c) 4 cis-3-hexenylacetate 62 ± 5^(d) 3,730 ± 79^(a)  1,884 ± 107^(b)   700 ± 143^(c) 5limonene 0^(b) 2,266 ± 146^(a) 2,230 ± 122^(a) 2,188 ± 137^(a) 6β-ocimene 0^(c) 4,000 ± 79^(a)  3,036 ± 116^(b) 0^(c) 7 linalool 0^(c) 456 ± 59^(b) 2,050 ± 73^(a) 1,964 ± 94^(a) 8 unknown 0^(c) 2,962 ±123^(a) 2,352 ± 210^(b) 2,962 ± 45^(a) 9 caryophyllene 75 ± 10^(d) 6,928± 787^(b) 8,380 ± 842^(a) 3,182 ± 200^(c) 10 α-humulene 0^(c) 1,844 ±136^(a) 1,811 ± 120^(a)   288 ± 42^(b) 11 β-farnesene 0^(c) 1,836 ±96^(a)  1,830 ± 52^(a)   284 ± 56^(b) Note: Volatiles were collected for24 h. ^(a)In order of elution during gas chromatography ^(b)Values(amount emitted) are mean ng amount ± SE of five replicates Means acrossthe same row followed by different letters are significantly different(P < 0.05, ANOVA)

PGPR strain INR-7 treated cotton plants released significantly moreα-pinene, β-pinene, β-myrcene, cis-3-hexenyl acetate, and β-ocimene thanBlend 8 or Blend 9 treated plants (Table 2, FIG. 1). Additionally,β-ocimene was not detected in Blend 9 (Table 2, FIG. 1). FIG. 2 showsthe GC profiles of the headspace volatiles emitted by the following fourtreatments: untreated (control) uninfested plants, untreated (control)H. virescens infested plants, PGPR Blend 9 treated uninfested plants,and PGPR Blend 9 treated H. virescens infested plants.

Identical peaks (28) were detected in extracts of untreated (control) H.virescens infested plants and PGPR Blend 9 treated H. virescens infestedplants (Table 3, FIG. 2). However, 10 peaks (components) were detectedin PGPR Blend 9 treated uninfested plants compared with only 3 peaksdetected in untreated (control) uninfested plants (FIG. 2).

TABLE 3 Composition of headspace volatiles emitted by untreated(control) uninfested cotton plants vs. untreated (control) H. virescensinfested plants, PGPR Blend 9 treated uninfested plants, or PGPR Blend 9treated H. virescens infested plants Untreated Untreated (control)(control) PGPR Blend 9 treated uninfested H. virescens PGPR Blend 9treated H. virescens infested ID Compound cotton plants infested plantsuninfested plants plants 1 cis-3-hexenal 0 39,740 ± 2985^(a) 0 38,844 ±3397^(a) 2 trans-2-hexenal 0 63,131 ± 2653^(a) 0 63,020 ± 2527^(a) 3cis-3-hexen-1-ol 0 15,720 ± 916^(a)  0 15,340 ± 1262^(a) 4trans-2-hexen-1-ol 0 68,602 ± 2774^(a) 0 68,802 ± 2451^(a) 5 α-pinene 58± 12^(c) 93,110 ± 1345^(a) 5,714 ± 519^(b)   95,110 ± 1081^(a) 6β-pinene 0 58,039 ± 4522^(a) 786 ± 132^(b) 57,839 ± 1606^(a) 7 myrcene 0120,239 ± 6930^(a)  864 ± 148^(b) 119,979 ± 6500^(a)  8 cis-3-hexenylacetate 0 161,450 ± 5000^(a)  700 ± 143^(b) 163,510 ± 4300^(a)  9trans-2-hexenyl acetate 0 98,814 ± 1892^(a) 0 99,270 ± 1504^(a) 10limonene 0 110,272 ± 3614^(a)  2,188 ± 137^(b)   110,059 ± 3460^(a)  11β-ocimene 0 120,177 ± 3147^(a)  0 120,466 ± 4200^(a)  12 linalool 62 ±16^(c) 18,343 ± 1704^(a) 1,964 ± 94^(b)   18,863 ± 1660^(a) 13 unknown 057,320 ± 2531^(a) 2,962 ± 45^(b)   60,720 ± 2100^(a) 144,8-dimethyl-1,3,7- 0 20,920 ± 2166^(a) 0 20,736 ± 2109^(a) nonatriene15 cis-3-hexenyl butyrate 0 106,285 ± 2136^(a)  0 108,725 ± 4628^(a)  16trans-2-hexenyl butyrate 0 88,170 ± 2420^(a) 0 90,730 ± 3256^(a) 17n-decanal 0 4,700 ± 541^(a) 0 4,900 ± 877^(a) 18 cis-3-hexenyl-2-methyl0 135,100 ± 6607^(a)  0 135,695 ± 6779^(a)  butyrate 19trans-2-hexenyl-2-methyl 0 128,350 ± 5055^(a)  0 126,950 ± 6136^(a) butyrate 20 indole 0 58,430 ± 2051^(a) 0 68,430 ± 1934^(a) 21 isobutyltiglate 0 15,700 ± 1139^(a) 0 15,500 ± 1028^(a) 22 2-hexenyl tiglate 06,700 ± 190^(a) 0 6,620 ± 97^(a)  23 cis-jasmone 0 55,811 ± 928^(a)  069,200 ± 1484^(a) 24 caryophyllene 75 ± 10 172,500 ± 6461^(a)  3,182 ±200^(c)   186,500 ± 6825^(b)  25 α-trans bergamotene 0 15,778 ± 832^(b) 0 17,578 ± 817^(a)  26 α-farnesene 0 38,145 ± 1754^(a) 288 ± 42^(b) 39,345 ± 1500^(a) 27 α-humulene 0 32,400 ± 1023^(a) 0 34,800 ± 994^(a) 28 β-farnesene 0 47,979 ± 870^(a)  0 52,439 ± 1072^(a) Note: Volatileswere collected for 24 h. ¹In order of elution during gas chromatography²Values (amount emitted) are mean ng amount ± SE of five replicateextractions Means across the same row followed by different letters aresignificantly different (P < 0.05, ANOVA).

Analysis of Cotton Root Growth.

Cotton root growth promotion resulting after PGPR treatment is shown inFIGS. 2, 3, and 4. Inoculation of cotton seeds with PGPR strains INR-7,Blend 8, and Blend 9, significantly promoted growth as compared to theuntreated control. Significant differences were recorded among thetreatments in root surface area (F_(3,7)=74.78, P<0.0001; FIG. 3), rootvolume (F_(3,7)=50.42, P<0.0001; FIG. 4), and root dry weight(F_(3,7)=28.07, P<0.0001; FIG. 5). In all cases, Blend 9-treated plantshad the highest root surface area, root volume, and root dry weight.INR-7 and Blend 8-treated plants also had significantly higher rootgrowth parameters than untreated plants (FIGS. 3, 4 and 5).

Four-Choice Olfactometer Bioassays with Parasitoids.

In the first experiment, significant differences were recorded in theresponse of female M. croceipes to the two treatments and two controls.Parasitoids were significantly (F_(3,7)=106.64, P<0.0001) more attractedto Blend 9-treated plants (69%) compared with INR-7 treated plants(29%), untreated (control) plants (0%), or blank control (empty chamber)(0%) (FIG. 6). Significant differences were also recorded among thetreatments (F_(3,12)=35.92, P<0.0001) in experiment 2, which wasdesigned to determine if PGPR treatment is as effective as caterpillarinfestation/damage (30 H. virescens caterpillars) in attractingparasitoids to plants. As expected, PGPR Blend 9-treated plants infestedwith 30 caterpillars (46%) and untreated (control) plants infested with30 caterpillars (41%) were highly attractive to parasitoids. However,parasitoids were more attracted to untreated (control) plants infestedwith 30 caterpillars (41%) than to uninfested PGPR Blend 9-treatedplants (13%) (FIG. 7), suggesting that PGPR treatment was not as potentas infestation with 30 caterpillars in attracting parasitoids. Theresults of the third experiment, in which a lower level of infestation(2 H. virescens caterpillars per plant) was tested, also showedsignificant differences among the treatments and controls(F_(3,12)=7.12, P=0.0053). The most attractive treatment was PGPR Blend9-treated plants infested with two caterpillars (58%). However,significantly more parasitoids were attracted to uninfested PGPR Blend 9treated plant (25%) compared with untreated (control) plants infestedwith two caterpillars (15%) (FIG. 8). These results showed that PGPRtreatment was at least as effective as low levels of caterpillar damagein attracting parasitoids to plants.

Discussion

These results show that plant growth-promoting rhizobacteria (PGPR)alter volatile organic compounds (VOCs) production in cotton plants. Thediscovery that PGPR alters the production of VOCs in cotton constitutesan unreported mechanism for the elicitation of plant volatile productionby rhizobacteria. All tested PGPR treatments (INR7, Blend 8 and Blend 9)elicited the emission of VOCs that were not detected in untreated cottonplants. Eleven components were detected in the headspace of PGPR-treatedplants. In the headspace of untreated plants, most of these compoundswere not detected or were detected in insignificant amounts (only threewere detected). In addition to altering VOC production, PGPR treatmentsalso led to cotton plant root growth promotion, with Blend 9 showing thehighest root growth promotion. PGPR have previously been reported topromote plant growth (including roots) in several plant species. Mostintriguingly, results from the four-choice olfactometer experiments showthat parasitoids were able to distinguish between PGPR treated anduntreated plants, preferring the former over the latter.

The major components detected in headspace collections of PGPR-treatedplants were: α-pinene, β-pinene, β-myrcene, cis-3-hexenyl acetate,limonene, β-ocimene, linalool, caryophyllene, α-humulene, andβ-farnesene (Table 2, FIG. 1). These compounds have been reported beforeto be constituents of blends of VOCs emitted from caterpillar damagedcotton plants (Loughrin et al. 1994, De Moraes et al. 1998, Ngumbi etal. 2009). However, unlike previous reports, the PGPR-induced blend ofVOCs is qualitatively different from VOCs emitted by caterpillar damagedplants (Table 3, FIG. 2). Differences in the quality of the blend ofVOCs are defined as differences in the presence of specific compounds inthe blend and/or ratio of the components. These results suggest thatsome VOCs, such as α-pinene, β-pinene, cis-3-hexenyl acetate, limonene,β-ocimene, linalool, caryophyllene, α-humulene, and β-farnesene may beelicited by PGPR. Previous studies have reported that VOC production inplants may be elicited by plant hormones (de Bruxelles and Roberts,2001, Thaler et al. 2002, Farmer et al. 2003, Ament et al. 2004),herbivore-derived elicitors (Mattiaci et al. 1995, Alborn et al. 1997,Spiteller and Boland, 2003), pathogens (Cardoza et al. 2002), wounding(Mithöfer et al. 2005), and heavy metals (Mithöfer et al. 2004). Thesefindings demonstrate that PGPR elicit the induction of VOCs and furtherstudies are warranted to understand the mechanisms by which treatment ofcotton plants with PGPR led to the release of VOCs that differ fromuntreated plants.

These data on cotton root analysis suggest that PGPR treatment enhancedcotton root growth. Increase in root weight growth as a result of PGPRtreatment has been recorded for other crops, including sweet basil(Ocimum basilicum L.) and tomato (Solanum lycopersicum L.) (Kloepper1992, Zehnder et al. 1997, Kloepper et al. 2004, Burkett-Cadena et al.2008, Banchio et al. 2009, Humberto et al. 2010). PGPR have been appliedto different crops for the purposes of growth enhancement and otherpositive effects in plants, such as seed emergence, tolerance todrought, and increase in weight of plant shoots and roots (Glick 1995,Kloepper et al. 2004, Kokalis-Burelle et al. 2006, Yildirim et al. 2006;van Loon, 2007). Humberto et al. (2010) showed that inoculation oftomato plants with growth promoting Bacillus subtilis led to tomato rootgrowth promotion and this was evident 3 weeks after inoculation. Thesefindings corroborate these results in which growth promotion of cottonroots was evident 2 weeks after inoculation. In addition to promotingroot growth, PGPR-treated plants enhance a plant's ability to defenditself from insects and pathogens by eliciting defensive responses, alsoknown as induced systemic resistance (ISR) (Kloepper et al. 2004) or byantibiosis (Zehnder et al. 2001). Some of the reported examples includereduced insect herbivory in cucumber Cucumis saliva (L.) (Zehnder et al.1997) and resistance to whitefly Bemicia tabaci (Hanafi et al. 2007).

The results of the behavioral experiments clearly show the ability ofthe specialist parasitic wasp, M. croceipes, to detect, distinguish andexploit the differences between PGPR treated versus untreated plants.Specifically, PGPR treated plants were highly attractive to parasitoids,with Blend 9 treated plants being the most attractive. Furtherevaluation demonstrated that Blend 9-treated but uninfested plants wereeven more attractive to parasitoids than untreated plants with lowlevels of caterpillar infestations (2 H. virescens caterpillars perplant). Volatile organic compounds (VOCs) emitted systematically byplants can act as host location cues for foraging parasitoids (Röse etal. 1998, De Moraes et al. 1998, Ngumbi et al. 2009). These resultsshowed that PGPR-treated plants were highly attractive to parasitoids ascompared to untreated plants. These findings could be attributed to theblend of VOCs being produced by the PGPR-treated plants that is absentin the headspace of untreated plants. These PGPR-induced compounds havebeen implicated in natural enemy attraction through behavioral studiesand antennal electrophysiological studies (Rose et al. 1998, Chen andFadamiro 2007, Ngumbi et al. 2009). These data clearly showed theability of the specialist parasitic wasp, M. croceipes, to detect,distinguish and exploit the differences between PGPR-treated versusuntreated plants.

Among the tested PGPR treatments, Blend 9-treated plants were the mostattractive to parasitoids. Interestingly, Blend 9-treated plantsconsistently did not release β-ocimene. Thus, the absence of 1-ocimenein the blend of VOCs emitted by Blend 9 treated plants might beresponsible for the enhanced attraction of M. croceipes to PGPR Blend9-treated plants. Previous studies have reported that parasitoids likeM. croceipes can detect and exploit qualitative and quantitativedifferences in blends of VOCs when searching for their herbivore hosts(De Moraes et al. 1998). In a related study investigating the impact ofPGPR on natural enemies of Myzus persicae (Hemiptera: Aphididae),Boutard-Hunt et al. (2009) reported that densities of natural enemieswere significantly higher in plots treated with PGPR as compared tountreated plots. By providing specific and reliable chemical signals,plants may acquire a competitive advantage in the recruitment ofherbivore natural enemies.

In summary, these results show that treatment of cotton plants withsingle strains or blends of several strains of PGPR (plantgrowth-promoting rhizobacteria) elicits changes in cotton plant VOCswith important consequences for foraging parasitoids. Together, theresults suggest that PGPR treatment could signal low levels ofcaterpillar damage necessary for attraction of parasitoids to plants,most likely via increased emission of HIPVs. These findings establish anew function for PGPR in mediating insect-plant and tri-trophicinteractions.

Further studies are needed to investigate if increased emission andinduction of VOCs by PGPR is a common phenomenon in multiple crops underdifferent ecological conditions. Additional studies are necessary totest if key natural enemy species in other cropping systems show similarresponse to PGPR-treated plants. If confirmed, results from such studieswill demonstrate that treatment of plants with PGPR may be a viablecomponent of integrated pest management of pests in manyagro-ecosystems.

Example 2 Effects of PGPR on the Egg-Laving Behavior of SpodopteraExigua

Abstract

Treating crops with plant growth-promoting rhizobacteria (PGPR) has beenshown to increase plant growth and enhance plant health in a variety ofways. Previously, these bacteria have been studied in models using onlyone to two strains of PGPR, limiting our understanding of how differentstrains may interact. Furthermore, little is known about the potentialeffects of PGPR on plant insect interactions. To determine the effectsof PGPR on the oviposition behavior of Spodoptera exigua on PGPR-treatedcotton plants, an egg-laying choice study was performed. The totalnumber of eggs and egg batches laid on cotton plants treated with PGPRversus untreated cotton plants were recorded. Here, Spodoptera exiguaexhibited an egg-laying preference on untreated cotton plants versusPGPR-treated cotton plants. No eggs were recorded on one of the testedPGPR treatments comprising Bacillus amyloliquefaciens.

Materials and Results

Two experiments were conducted to test different PGPR blends/strains. InExperiment 1, cotton plant seeds were treated with 1 ml of threedifferent aqueous spore seed preparations (Blend 8, Blend 9, and INR-7,see Example 1) at a concentration of 10⁷ cfu/ml. In Experiment 2, PGPRstrains ABU 288 (Bacillus simplex) and MBI 600 (Bacillus subtilis) weretested. An untreated control was included for both Experiments.

Female and male Spodoptera exigua were allowed to mate. Males weremarked for separation later, and thirty mated females were separatedfrom the males. For each of 8 replicates, 30 mated female Spodopteraexigua were caged overnight for a 14 hour period. The cage (42″×42″×32″tall) was placed in a dark room and each of the four corners had onecotton plant (4-5 weeks old) of each of the four treatments,respectively. Plant stems were placed 80 cm apart, and plants wererotated between replicates. After 14 hours, the number of egg masses andtotal number of eggs per plant were counted. Results are presented inTable 4 and FIG. 9 for Experiment 1, and in Table 5 for Experiment 2.

TABLE 4 Egg-laying of Spodoptera exigua on PGPR-treated (INR7, Blend 8and Blend 9) versus untreated cotton plants Number of replicates Totalduring which treatment number of Total number of egg plant had eggs laidon it Treatment eggs laid batches laid (out of 8) Control 491 32 8 INR-7151 7 4 Blend 8 341 22 6 Blend 9 0 0 0

TABLE 5 Egg-laying of Spodoptera exigua on PGPR-treated (strains MBI 600or ABU 288) versus untreated cotton plants Total number of Total numberof egg Treatment eggs laid batches laid Control 105 3 MBI 600 0 0 ABU288 0 0

The results of both experiments illustrate a trend whereby Spodopteraexigua preferred to lay their eggs on untreated cotton plants versusPGPR-treated cotton plants. No eggs were laid on PGPR Blend 9(Experiment 1) and strains MBI 600 and ABU 288 (Experiment 2).

Headspace volatiles were collected for plants in Experiment 2. As forplants treated with Blend 8, Blend 9, and INR-7 (see Example 1, FIG. 1),induction of VOCs by plants treated with strains MBI 600 and ABU 288versus control plants was observed (FIG. 10). Quantitative andqualitative differences in headspace volatiles collected from cottontreated with either strain compared to untreated cotton plants wereobserved. The peaks induced by strains MBI 600 and ABU 288 versuscontrol likely are small molecular weight terpenoid compounds such asmonoterpenes and sesquiterpenes.

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It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein. The terms and expressions whichhave been employed are used as terms of description and not oflimitation, and there is no intention in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention. Thus, itshould be understood that although the present invention has beenillustrated by specific embodiments and optional features, modificationand/or variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention.

Citations to a number of patent and non-patent references are madeherein. The cited references are incorporated by reference herein intheir entireties. In the event that there is an inconsistency between adefinition of a term in the specification as compared to a definition ofthe term in a cited reference, the term should be interpreted based onthe definition in the specification.

1.-30. (canceled)
 31. A composition for treating a plant or seeds of aplant, the composition formulated as a treatment composition in whichare combined: (a) an isolated Bacillus bacteria selected from the groupconsisting of: (i) Bacillus amyloliquefaciens selected from the groupconsisting of Bacillus amyloliquefaciens strain AP-136 (NRRL B-50614),Bacillus amyloliquefaciens strain AP-188 (NRRL B-50615), Bacillusamyloliquefaciens strain AP-218 (NRRL B-50618), Bacillusamyloliquefaciens strain AP-219 (NRRL B-50619), and Bacillusamyloliquefaciens strain AP-295 (NRRL B-50620); (ii) Bacillus mojavensisstrain AP-209 (NRRL B-50616); (iii) Bacillus solisalsi strain AP-217(NRRL B-50617); (iv) Bacillus simplex strain ABU 288 (NRRL B-50340); and(v) mixtures thereof; and (b) a suitable carrier, the compositioninducing production of one or more volatile organic compounds (VOCs) bya plant that has been treated with the composition or that is grown fromseed that has been treated with the composition.
 32. The compositionaccording to claim 31, wherein the bacteria are selected from a groupconsisting of Bacillus amyloliquefaciens strain AP-136 (NRRL B-50614),Bacillus amyloliquefaciens strain AP-188 (NRRL B-50615), Bacillusamyloliquefaciens strain AP-218 (NRRL B-50618), Bacillusamyloliquefaciens strain AP-219 (NRRL B-50619), and Bacillusamyloliquefaciens strain AP-295 (NRRL B-50620).
 33. The compositionaccording to claim 31, wherein the bacteria are Bacillus mojavensisstrain AP-209 (NRRL B-50616).
 34. The composition according to claim 31,wherein the bacteria are Bacillus solisalsi strain AP-217 (NRRLB-50617).
 35. The composition according to claim 31, wherein thebacteria are Bacillus simplex strain ABU 288 (NRRL B-50340).
 36. Thecomposition according to claim 31, wherein the carrier is selected fromthe group consisting of peat, wheat, bran, vermiculite, and pasteurizedsoil.
 37. The composition according to claim 31, wherein the one or moreVOCs comprise one or more compounds selected from a group consisting ofalpha-pinene, beta-pinene, beta-myrcene, cis-3-hexenyl acetate,limonene, beta-ocimene, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene,methyl salicylate, decanal, cis-jasmone, caryophyllene, alpha-humulene,beta-farnesene, and mixtures thereof.
 38. The composition according toclaim 31, wherein the insect is an herbivore and the one or more VOCsreduce egg-laying of the insect on the plant.
 39. The compositionaccording to claim 31, wherein the insect is an herbivore and the one ormore VOCs reduce feeding of the insect on the plant.
 40. The compositionaccording to claim 31, wherein the insect is a predator or a parasitoidand the one or more VOCs attract the predator or the parasitoid to theplant.
 41. A plant treated with the composition of claim
 31. 42. Theplant of claim 41, wherein the plant is selected from the groupconsisting of alfalfa, rice, barley, rye, cotton, sunflower, peanut,corn, potato, sweet potato, bean, pea, lentil chicory, lettuce, endive,cabbage, brussel sprout, beet, parsnip, turnip, cauliflower, broccoli,turnip, radish, spinach, onion, garlic, eggplant pepper, celery, carrot,squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus,strawberry, grape, raspberry, pineapple, soybean, canola, oil seed rape,spring wheat, winter wheat, tobacco, tomato, sorghum, and sugarcane. 43.Plant seeds treated with the composition of claim
 31. 44. The plantseeds of claim 43, wherein the plant seeds are of a plant selected fromthe group consisting of alfalfa, rice, barley, rye, cotton, sunflower,peanut, corn, potato, sweet potato, bean, pea, lentil chicory, lettuce,endive, cabbage, brussel sprout, beet, parsnip, turnip, cauliflower,broccoli, turnip, radish, spinach, onion, garlic, eggplant pepper,celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon,citrus, strawberry, grape, raspberry, pineapple, soybean, canola, oilseed rape, spring wheat, winter wheat, tobacco, tomato, sorghum, andsugarcane.
 45. A method of treating a plant, the method comprisingadministering the composition of claim 31 to the plant or to soilsurrounding the plant.
 46. The method according to claim 45, wherein theplant is selected from the group consisting of alfalfa, rice, barley,rye, cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea,lentil chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip,turnip, cauliflower, broccoli, turnip, radish, spinach, onion, garlic,eggplant pepper, celery, carrot, squash, pumpkin, zucchini, cucumber,apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple,soybean, canola, oil seed rape, spring wheat, winter wheat, tobacco,tomato, sorghum, and sugarcane.
 47. The method according to claim 45,wherein the method reduces egg-laying of an insect on the plant.
 48. Themethod according to claim 45, wherein the method reduces feeding of aninsect on the plant.
 49. The method according to claim 45, wherein themethod attracts a predator insect or parasitoid to the plant.
 50. Amethod of treating a plant seed, the method comprising treating theplant seed with the composition of claim 31.